Enterohemorrhagic E. coli (EHEC) is a deadly food borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. The main reservoir for EHEC is cattle herds. EHEC colonizes the recto anal junction (RAJ) of cattle, forming attaching and effacing (AE) lesions employing the locus of enterocyte effacement (LEE) genes. Additionally, EHEC also requires the glutamate acid resistance (gad) system to survive the acidic stomachs of these animals and successfully colonize the RAJ. Coordinated expression of the LEE and gad genes is achieved through the SdiA transcription factor. SdiA is a homolog of LuxR, which is a transcription factor involved in bacterial quorum sensing (QS), functioning as a receptor for the bacterial autoinducer acyl-homoserine lactone (AHL). Although E. coli (including EHEC) does not produce AHLs, they have the ability to respond to these signals through SdiA. The PI reported that SdiA senses AHLs produced by the rumminal microbiota to repress expression of the LEE genes, which is an unnecessary expense of energy in this gastrointestinal (GI) compartment, and activate expression of the gad genes to prime EHEC's acid resistance before it reaches the acidic stomachs. This coordinated SdiA-mediated gene regulation is necessary for EHEC's efficient colonization of cattle. Most LuxR-type transcription factors require the AHL signal as a folding switch. Although it was initially proposed that this scenario was also the case for SdiA, there is mounting evidence that SdiA can regulate transcription of several genes, including the gad genes, in the absence of AHLs. We solved the structures of SdiA bound to two AHLs, oxo-C6-homoserine lactone (HSL), and oxo-C8-HSL, as well as in the absence of AHL. The crystal structures of both AHL-bound forms of SdiA overlay perfectly with each other. However, there is a discreet structural change in the overall structure of the SdiA AHL-bound protein compared to no AHL, which is more pronounced in the helix-turn-helix (HTH) DNA binding domain of this protein. This structural change confers higher DNA binding affinity to the AHL bound form of this protein. These structural studies also revealed that EHEC produces an SdiA endogenous ligand identified as 1-Octanoyl-rac-glycerol, a monoacylglycerol that constitutes one of the building blocks of triacylglycerols. Accordingly the Specific Aims of this application are: Specific Aim 1. Identify te synthetic pathway of the SdiA endogenous ligand. Specific Aim 2. Define the requirements for different signal recognition and function within the SdiA ligand-binding pocket. Specific Aim 3. Determine the molecular mechanism of SdiA- dependent gad regulation.